NO753291L - - Google Patents
Info
- Publication number
- NO753291L NO753291L NO753291A NO753291A NO753291L NO 753291 L NO753291 L NO 753291L NO 753291 A NO753291 A NO 753291A NO 753291 A NO753291 A NO 753291A NO 753291 L NO753291 L NO 753291L
- Authority
- NO
- Norway
- Prior art keywords
- methanol
- iii
- antibiotics
- strain
- see
- Prior art date
Links
- 239000003242 anti bacterial agent Substances 0.000 claims abstract description 26
- 229940088710 antibiotic agent Drugs 0.000 claims abstract description 26
- 238000000034 method Methods 0.000 claims abstract description 17
- 239000000126 substance Substances 0.000 claims description 17
- 241000133685 Aspergillus rugulosus Species 0.000 claims description 9
- 235000015097 nutrients Nutrition 0.000 claims description 8
- 238000000855 fermentation Methods 0.000 claims description 5
- 230000004151 fermentation Effects 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 231100000219 mutagenic Toxicity 0.000 claims description 2
- 230000003505 mutagenic effect Effects 0.000 claims description 2
- 230000005855 radiation Effects 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 abstract description 8
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 99
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 24
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 18
- 241001465754 Metazoa Species 0.000 description 17
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 17
- 239000000741 silica gel Substances 0.000 description 17
- 229910002027 silica gel Inorganic materials 0.000 description 17
- 208000015181 infectious disease Diseases 0.000 description 16
- 241000222122 Candida albicans Species 0.000 description 14
- 239000000243 solution Substances 0.000 description 14
- 229940095731 candida albicans Drugs 0.000 description 13
- 238000000354 decomposition reaction Methods 0.000 description 12
- 230000008018 melting Effects 0.000 description 12
- 238000002844 melting Methods 0.000 description 12
- 238000010828 elution Methods 0.000 description 11
- 239000010410 layer Substances 0.000 description 11
- 244000052616 bacterial pathogen Species 0.000 description 10
- 238000011534 incubation Methods 0.000 description 10
- 239000000203 mixture Substances 0.000 description 10
- 239000000843 powder Substances 0.000 description 10
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 10
- 238000012360 testing method Methods 0.000 description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- 238000005984 hydrogenation reaction Methods 0.000 description 9
- 241000894007 species Species 0.000 description 9
- 238000001228 spectrum Methods 0.000 description 9
- 229920001817 Agar Polymers 0.000 description 8
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 8
- 239000008272 agar Substances 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 7
- 238000004587 chromatography analysis Methods 0.000 description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 6
- 241000700159 Rattus Species 0.000 description 6
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 description 6
- 239000002674 ointment Substances 0.000 description 6
- CZDYPVPMEAXLPK-UHFFFAOYSA-N tetramethylsilane Chemical compound C[Si](C)(C)C CZDYPVPMEAXLPK-UHFFFAOYSA-N 0.000 description 6
- 238000002211 ultraviolet spectrum Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 238000005160 1H NMR spectroscopy Methods 0.000 description 5
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 5
- 230000037396 body weight Effects 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 210000003608 fece Anatomy 0.000 description 5
- 238000002329 infrared spectrum Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 229960005322 streptomycin Drugs 0.000 description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 230000007062 hydrolysis Effects 0.000 description 4
- 238000006460 hydrolysis reaction Methods 0.000 description 4
- 229960000988 nystatin Drugs 0.000 description 4
- VQOXZBDYSJBXMA-NQTDYLQESA-N nystatin A1 Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/CC/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 VQOXZBDYSJBXMA-NQTDYLQESA-N 0.000 description 4
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 3
- 206010064899 Vulvovaginal mycotic infection Diseases 0.000 description 3
- 239000006227 byproduct Substances 0.000 description 3
- 239000003054 catalyst Substances 0.000 description 3
- SIHHLZPXQLFPMC-UHFFFAOYSA-N chloroform;methanol;hydrate Chemical compound O.OC.ClC(Cl)Cl SIHHLZPXQLFPMC-UHFFFAOYSA-N 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- FAUOJMHVEYMQQG-HVYQDZECSA-N echinocandin B Chemical compound C1([C@H](O)[C@@H](O)[C@H]2C(=O)N[C@H](C(=O)N3C[C@H](C)[C@H](O)[C@H]3C(=O)N[C@H](O)[C@H](O)C[C@@H](C(N[C@H](C(=O)N3C[C@H](O)C[C@H]3C(=O)N2)[C@@H](C)O)=O)NC(=O)CCCCCCC\C=C/C\C=C/CCCCC)[C@@H](C)O)=CC=C(O)C=C1 FAUOJMHVEYMQQG-HVYQDZECSA-N 0.000 description 3
- 235000013399 edible fruits Nutrition 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 230000002538 fungal effect Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 3
- 238000009806 oophorectomy Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 241000228212 Aspergillus Species 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 206010022678 Intestinal infections Diseases 0.000 description 2
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 238000005273 aeration Methods 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- TZCXTZWJZNENPQ-UHFFFAOYSA-L barium sulfate Chemical compound [Ba+2].[O-]S([O-])(=O)=O TZCXTZWJZNENPQ-UHFFFAOYSA-L 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000006787 czapek-dox agar Substances 0.000 description 2
- 239000008121 dextrose Substances 0.000 description 2
- 239000003651 drinking water Substances 0.000 description 2
- 235000020188 drinking water Nutrition 0.000 description 2
- 230000008030 elimination Effects 0.000 description 2
- 238000003379 elimination reaction Methods 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 230000012173 estrus Effects 0.000 description 2
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000002523 gelfiltration Methods 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 210000000936 intestine Anatomy 0.000 description 2
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 2
- 235000020778 linoleic acid Nutrition 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000012299 nitrogen atmosphere Substances 0.000 description 2
- 239000003883 ointment base Substances 0.000 description 2
- 229910052763 palladium Inorganic materials 0.000 description 2
- 239000003208 petroleum Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 238000013207 serial dilution Methods 0.000 description 2
- 239000011877 solvent mixture Substances 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000000052 vinegar Substances 0.000 description 2
- 235000021419 vinegar Nutrition 0.000 description 2
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 description 1
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- DKPFZGUDAPQIHT-UHFFFAOYSA-N Butyl acetate Natural products CCCCOC(C)=O DKPFZGUDAPQIHT-UHFFFAOYSA-N 0.000 description 1
- 208000032544 Cicatrix Diseases 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 229920004011 Macrolon® Polymers 0.000 description 1
- 206010065764 Mucosal infection Diseases 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- AFCARXCZXQIEQB-UHFFFAOYSA-N N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CCNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 AFCARXCZXQIEQB-UHFFFAOYSA-N 0.000 description 1
- 238000011785 NMRI mouse Methods 0.000 description 1
- 241000364057 Peoria Species 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 241000700157 Rattus norvegicus Species 0.000 description 1
- 239000006159 Sabouraud's agar Substances 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- 201000007096 Vulvovaginal Candidiasis Diseases 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- -1 aliphatic alcohols Chemical class 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000009903 catalytic hydrogenation reaction Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- VNFPBHJOKIVQEB-UHFFFAOYSA-N clotrimazole Chemical compound ClC1=CC=CC=C1C(N1C=NC=C1)(C=1C=CC=CC=1)C1=CC=CC=C1 VNFPBHJOKIVQEB-UHFFFAOYSA-N 0.000 description 1
- 229960004022 clotrimazole Drugs 0.000 description 1
- 238000012505 colouration Methods 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- IJKVHSBPTUYDLN-UHFFFAOYSA-N dihydroxy(oxo)silane Chemical compound O[Si](O)=O IJKVHSBPTUYDLN-UHFFFAOYSA-N 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 229960005309 estradiol Drugs 0.000 description 1
- 229930182833 estradiol Natural products 0.000 description 1
- 239000002024 ethyl acetate extract Substances 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 238000012262 fermentative production Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 229960000907 methylthioninium chloride Drugs 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 239000006916 nutrient agar Substances 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229940093430 polyethylene glycol 1500 Drugs 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920001451 polypropylene glycol Polymers 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000012449 sabouraud dextrose agar Substances 0.000 description 1
- 231100000241 scar Toxicity 0.000 description 1
- 230000037387 scars Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000008159 sesame oil Substances 0.000 description 1
- 235000011803 sesame oil Nutrition 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- QGMRQYFBGABWDR-UHFFFAOYSA-N sodium;5-ethyl-5-pentan-2-yl-1,3-diazinane-2,4,6-trione Chemical compound [Na+].CCCC(C)C1(CC)C(=O)NC(=O)NC1=O QGMRQYFBGABWDR-UHFFFAOYSA-N 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000001117 sulphuric acid Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000002344 surface layer Substances 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 238000001665 trituration Methods 0.000 description 1
- 229960001005 tuberculin Drugs 0.000 description 1
- 210000001215 vagina Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/062—Ascomycota
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07G—COMPOUNDS OF UNKNOWN CONSTITUTION
- C07G11/00—Antibiotics
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Mycology (AREA)
- General Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Animal Behavior & Ethology (AREA)
- Alternative & Traditional Medicine (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Botany (AREA)
- Epidemiology (AREA)
- Biotechnology (AREA)
- Medical Informatics (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Engineering & Computer Science (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
- Compounds Of Unknown Constitution (AREA)
- Saccharide Compounds (AREA)
Abstract
Fremgangsmåte for fremstilling av nye_antibiotika.Procedure for the preparation of new antibiotics.
Description
Foreliggende oppfinnelse vedrører en fremgangsmåte for fremstilling av nye antibiotika, som i det følgende betegnes med SL 7810/F, The present invention relates to a method for the production of new antibiotics, which is hereinafter referred to as SL 7810/F,
SL 7810/F-II og SL 7810/F-III, samt deres tetrahydroderivater.SL 7810/F-II and SL 7810/F-III, as well as their tetrahydro derivatives.
Det særegne ved fremgangsmåten i henhold til oppfinnelsen for fremstilling av de nye antibiotika og deres tetrahydroderivater, The peculiarity of the method according to the invention for the production of the new antibiotics and their tetrahydroderivatives,
er atis that
a) en stamme av skekten aspergillus rugulosus Thorn & Raper dyrkes i nærvær av et næringsmedium, eller b) for fremstilling av tetrahydroderivatene av disse antibiotika underkastes de en hydrogenering. a) a strain of the genus Aspergillus rugulosus Thorn & Raper is grown in the presence of a nutrient medium, or b) for the production of the tetrahydroderivatives of these antibiotics, they are subjected to hydrogenation.
De nye antibiotika og deres tetrahydroderivater har følgende karakteristika: SL 7810/ F The new antibiotics and their tetrahydroderivatives have the following characteristics: SL 7810/ F
Fargeløst amorft pulver med smeltepunkt 160 - 163°C (spalting); Colorless amorphous powder with melting point 160 - 163°C (decomposition);
/"o7p° 48,3° '(c = 0,806 i;;metanol)./"o7p° 48.3°' (c = 0.806 in;; methanol).
Analyse: Funnet C 57,3 H 7,9 N 9,2 0 25,2 %Analysis: Found C 57.3 H 7.9 N 9.2 0 25.2%
Molvekt (bestemt osmometrisk i etanol): 1190Molar weight (determined osmometrically in ethanol): 1190
UV^spektrum i metanol : Amaks= 194 nm, log £' = 1,98UV spectrum in methanol: Amax= 194 nm, log £' = 1.98
(se fig. 1) 276 nm, log = 0,14(see Fig. 1) 276 nm, log = 0.14
IR-spektrum i nujol (se fig. 2)IR spectrum in nujol (see fig. 2)
<->1H-NMR i DMSO (100 MHz, tetrametylsilan som intern standard)<->1H-NMR in DMSO (100 MHz, tetramethylsilane as internal standard)
/"se fig. 3/./"see fig. 3/.
Hydrolysen av SL 7810/F med 6 N saltsyre (4 timer ved 110°C under N^-atmosfære) gir som hydrolyseprodukt blant annet linolsyre. The hydrolysis of SL 7810/F with 6 N hydrochloric acid (4 hours at 110°C under a N 2 atmosphere) gives, among other things, linoleic acid as a hydrolysis product.
(Løselighet, stabilitet, ty-nnskik tkromatogram og MIC-verdier se nedenfor). (Solubility, stability, thin-phase chromatogram and MIC values see below).
SL 7810/ F- IISL 7810/F-II
Fargeløst amorft pulver med smeltepunkt 158 - 160°C (spalting); Colorless amorphous powder with melting point 158 - 160°C (decomposition);
/a7p° =^7,9° (c = 0,81 i metanol)./α7p° =^7.9° (c = 0.81 in methanol).
Analyse: Funnet C 58,5 H 8,0 N 9,3 0 23,8 %Analysis: Found C 58.5 H 8.0 N 9.3 0 23.8%
Mol vekt (bestemt osmometrisk i metanol): 1018Molar weight (determined osmometrically in methanol): 1018
UV-spektrum i metanol: Å-maks 193 nm, log = 1,95UV spectrum in methanol: Å-max 193 nm, log = 1.95
(se fig. 4) 278 nm, log^<*>0,18 (see Fig. 4) 278 nm, log^<*>0.18
TR-spektrum i nujol (se fig. 5).TR spectrum in nujol (see fig. 5).
<\>H-NMR-spektrum i DMSO (100 MHz, tetrametylsilan som intern standard) /se fig. 6/. <\>H-NMR spectrum in DMSO (100 MHz, tetramethylsilane as internal standard) / see fig. 6/.
(Løselighet, stabilitet, tynnskiktkromatogram og MIC-verdier se nedenfor.) (Solubility, stability, thin layer chromatogram and MIC values see below.)
SL 7810/F-IIXSL 7810/F-IIX
Fargeløst amorft.pulver med smeltepunkt 164 - 168°C (spalting); Colorless amorphous powder with melting point 164 - 168°C (decomposition);
/a7p° b -53,0° (c = 1,00 i metanol). /α7p° b -53.0° (c = 1.00 in methanol).
Analyse: Funnet C 61,1 H 8,0 N 9,0 0 22,0 % Analysis: Found C 61.1 H 8.0 N 9.0 0 22.0%
UV-spektrum i metanol: /^maks 193 nm, log £' = 1,98 (se fig. 7) 278 nm, log = 0,24 UV spectrum in methanol: /^max 193 nm, log £' = 1.98 (see Fig. 7) 278 nm, log = 0.24
IR-spektrum i nujol (se fig. 8).IR spectrum in nujol (see fig. 8).
H-NMR-spektrum'i DMSO (100 MH&, tetrametyl sil an som intern standard) /se fig. 9/. H-NMR spectrum in DMSO (100 MH&, tetramethyl silane as internal standard) /see fig. 9/.
(Løselighet, stabilitet, tynnskiktkromatogram og MIC-verdier se nedenfor). (Solubility, stability, thin layer chromatogram and MIC values see below).
Tetrahydro- SL 7810/ FTetrahydro- SL 7810/ F
Fargeløst amorft pulver med smeltepunkt 173 - 178°C (spalting); etter krystal1isering fra etanol-vann (95 : 5) smeltepunkt 190 - 205°C / 210 - 212°C (spalting); Colorless amorphous powder with melting point 173 - 178°C (decomposition); after crystallization from ethanol-water (95 : 5) melting point 190 - 205°C / 210 - 212°C (decomposition);
A7d° = -47>8° (c = 0,984 i metanol).Δ7d° = -47>8° (c = 0.984 in methanol).
Analyse: Funnet C 56,8 H 8,0 N 9,0 0 25,5 %Analysis: Found C 56.8 H 8.0 N 9.0 0 25.5%
UV-spektrum i metanol: ^ ma)<;s 194 nm, log £' = 1,85UV spectrum in methanol: λ ma)<;s 194 nm, log £' = 1.85
(se fig. 10) 277 nm, log =0,09(see Fig. 10) 277 nm, log =0.09
227 (skulder)227 (shoulder)
log =1,00 log = 1.00
IR-spektrum i nujol (se fig. 11)IR spectrum in nujol (see fig. 11)
H-NMR-spektrum i DMSO (100 MHz, tetrametylsilah som intern standard ) £se fig. 127. H-NMR spectrum in DMSO (100 MHz, tetramethylsilah as internal standard) £see fig. 127.
<13>C-NMR-spektrum i CD^OD med tetrametylsil an. som intern standard (se tabell 1) <13>C-NMR spectrum in CD^OD with tetramethylsil an. as internal standard (see table 1)
(Løselighet, stabilitét, tynnskiktkromatogram og MIC-verdier se nedenfor). (Solubility, stability, thin layer chromatogram and MIC values see below).
Hydrolysen av tetrahydro-SL 7810/F med 6 N saltsyre (4 timer ved 110°C under N^-atmosfære) gir som hydrolyseprodukt blandt annet stearinsyré. The hydrolysis of tetrahydro-SL 7810/F with 6 N hydrochloric acid (4 hours at 110°C under a N 2 atmosphere) gives, among other things, stearic acid as a hydrolysis product.
Tetrahydro- SL 7810/ F- IITetrahydro- SL 7810/ F- II
Fargeløst amorft pulver med smeltepunkt 160 - 163°C ( spalting); /o7d° = -41,4° (c « 0,823 i metanol). Colorless amorphous powder with melting point 160 - 163°C (decomposition); /o7d° = -41.4° (c « 0.823 in methanol).
Analyse: Funnet C 58,7 H 8,1 N 9,6 0 23,7 %Analysis: Found C 58.7 H 8.1 N 9.6 0 23.7%
UV-spektrum i metanol ^ maks<1>94 nm, log £, ' = 1,90UV spectrum in methanol ^max <1>94 nm, log £, ' = 1.90
(se fig. 13) 278 nm, log £, ' = 0,25(see Fig. 13) 278 nm, log £, ' = 0.25
227 (skulder)227 (shoulder)
log £' = 1,07 log £' = 1.07
IR-spektrum i nujol (se fig. 14)IR spectrum in nujol (see fig. 14)
\H^NMR-spektrum i DMSO (90 MHz, tetrametylsil.an -som intern standard) /se fig. 15.7. \H^NMR spectrum in DMSO (90 MHz, tetramethylsilan - as internal standard) / see fig. 15.7.
13 13
C-NMR-spektrum i CD^OD med tetrametyls.ilan som intern standard (se tabell 2). C-NMR spectrum in CD^OD with tetramethylsilane as internal standard (see table 2).
(Løselighet, stabilitet, tynnskiktkromatogram og MIC-verdier se nedenfor). (Solubility, stability, thin layer chromatogram and MIC values see below).
Tetrahydro- SL 7810/ F- III Fargeløst amorft pulver med smeltepunkt 175 - 180°C (spalting); /bc7p°= -41,5° (c = 1,094 i metanol). Tetrahydro- SL 7810/ F- III Colorless amorphous powder with melting point 175 - 180°C (decomposition); /bc7p°= -41.5° (c = 1.094 in methanol).
Analyse: Funnet C 60,5 H8,5 N9,2 0 21,9 %-Analysis: Found C 60.5 H8.5 N9.2 0 21.9%-
UV-spektrum i metanol: A maks 193 nm, log £11,93UV spectrum in methanol: A max 193 nm, log £11.93
(se fig. 16) 278 nm, log = 0,19(see Fig. 16) 278 nm, log = 0.19
224 nm, log 1,01 (skulder) 224 nm, log 1.01 (shoulder)
IR-spektrum i nujol (se fig. 17)..IR spectrum in nujol (see fig. 17)..
H-NMR-spektrum i DMSO (100 MHz, tetrametylsilan som intern standard) /se fig. 18J7. H-NMR spectrum in DMSO (100 MHz, tetramethylsilane as internal standard) / see fig. 18J7.
(Løselighet, stabilitet, tynnskiktkromatogram og MIC-verdier se nedenfor). (Solubility, stability, thin layer chromatogram and MIC values see below).
LøselighetSolubility
Antibiotikan SL 7810/F, SL 7810/F-II og SL 7810/F-III såvel som deres tetrahydroderivater er ved romtemperatur lett løselige i metanol og etanol , tungt løselige i kloroform, eter, petroleter, heksan og praktisk uoppløselig i vann. The antibiotics SL 7810/F, SL 7810/F-II and SL 7810/F-III as well as their tetrahydroderivatives are at room temperature easily soluble in methanol and ethanol, poorly soluble in chloroform, ether, petroleum ether, hexane and practically insoluble in water.
StabilitetStability
De nye antibiotika SL 78.10/F, SL 7810/F-II og SL 7810/F-III er i nærvær av.lys og spesielt i luften lite stabile og spaltes. Spaltingen merkes også ved oppvarming over 50°C. Hydrogeneringen til tetrahydroderivatene eliminerer i stor utstrekning ustabiliteten. The new antibiotics SL 78.10/F, SL 7810/F-II and SL 7810/F-III are unstable in the presence of light and especially in air and decompose. The splitting is also noticeable when heated above 50°C. The hydrogenation to the tetrahydro derivatives largely eliminates the instability.
Karakteristisk for allé tre antibiotika og deres tetrahydroderivater er ustabiliteten under basiske betingelser. Ved en pH over 8 opptrer omdannelse til spaltingsprodukter som biologisk bare er svakt aktive henhv. ikke lenger er aktive. Characteristic of all three antibiotics and their tetrahydroderivatives is their instability under basic conditions. At a pH above 8, conversion to cleavage products occurs which are biologically only weakly active or are no longer active.
Tynnskik tkromatogramThin layer chromatogram
Analyse skjer på kiselgel merk 60-plater (0,25 mm skikttykkelse). Med de etterfølgende elueringsmidler kan følgende R^-verdier konstateres: Analysis takes place on silica gel mark 60 plates (0.25 mm layer thickness). With the following eluents, the following R^ values can be determined:
Antibiotikan SL 7810/F, SL 7810/F-II og SL 7810/F-III viser ved denne bestemmelser de samme R^-verdier som deres tetrahydroderivater. The antibiotics SL 7810/F, SL 7810/F-II and SL 7810/F-III show in this determination the same R^ values as their tetrahydro derivatives.
Ved fremkalling med en 0,2% Ce( SO^)2~løsning i 50% svovelsyre som dusjreagens kan følgende flekker (etter oppvarming ved, 130°C) påvises: When developing with a 0.2% Ce(SO^)2~ solution in 50% sulfuric acid as a shower reagent, the following spots (after heating at, 130°C) can be detected:
For deteksjon av antibiotikan SL 7810/F, SL 7810/F-II og SL 7810/F-III kan også joddamp anvendes. Iodine vapor can also be used for detection of the antibiotics SL 7810/F, SL 7810/F-II and SL 7810/F-III.
Skjelningen mellom de tre antibiotika SL 7810/F, SL 7810/F-II og SL 7810/F-III fra deres tetrahydroderivater kan også skje tynnskikt-kromatografisk på kiselgel -GF 254-plater (0,3 mm skikttykkelse), idet kiselgelen er blandet med 3% AgNO^. Med kloroform-metanol-vann (70 : 25 : 5) som elueringsmiddel kan det med 40ifsubstans konstateres følgende R^-verdier: The separation between the three antibiotics SL 7810/F, SL 7810/F-II and SL 7810/F-III from their tetrahydroderivatives can also be done by thin-layer chromatography on silica gel -GF 254 plates (0.3 mm layer thickness), as the silica gel is mixed with 3% AgNO^. With chloroform-methanol-water (70 : 25 : 5) as eluent, the following R^-values can be determined with 40ifsubstance:
For påvisning av substansene dusjes med vanillin-svovelsyre. To detect the substances, shower with vanillin-sulphuric acid.
Ved oppvarming til 120 - 130°C opptrer gulbrune fargeflekker for tetrahydroforbindelsene. De ikke-hydrogenerte forbindelser viser en rødaktig farging. When heated to 120 - 130°C, yellow-brown color spots appear for the tetrahydro compounds. The non-hydrogenated compounds show a reddish colouration.
Fremgangsmåten a) lar seg gjennomføre på for fermenterende fremstilling av antibiotika kjente måter. En kultur derav ble deponert i United States Department of Agriculture (Northern Utilization Research and Development Diviåon), Peoria, 111., USA og står til disposisjon for offentligheten under betegnelsen NRRL 8039. Foretrukket anvendes stammen NRRL 8039. Method a) can be carried out in known ways for fermentative production of antibiotics. A culture thereof was deposited in the United States Department of Agriculture (Northern Utilization Research and Development Division), Peoria, 111., USA and is available to the public under the designation NRRL 8039. The strain NRRL 8039 is preferably used.
Imidlertid kan også stammer anvendes som er utvunnet fra utgangsstammen aspergillus rugulosus Thorn & Raper ved behandling med mutagene substanser eller stråler eller ved seleksjon. However, strains can also be used which have been extracted from the starting strain aspergillus rugulosus Thorn & Raper by treatment with mutagenic substances or radiation or by selection.
Karakteristikum for stammen NRRL 8039Characteristics of strain NRRL 8039
Den nye stamme NRRL 8039 av aspergillus rugulosus Thorn & Raper ble isolert fra en jordprøve isolert på Jaum-passet i Sveits og lar seg på grunn av sine morftologiske trekk ved hjelp av aspergillus-monografien av K.B. Raper & D.I. Fennell (THE GENUS ASPERGILLUS: The Williams & Wilkings Company Baltimore, 1965, side 686) inordne The new strain NRRL 8039 of aspergillus rugulosus Thorn & Raper was isolated from a soil sample isolated on the Jaum Pass in Switzerland and, due to its morphological features, allows the aspergillus monograph by K.B. Raper & D.I. Fennell (THE GENUS ASPERGILLUS: The Williams & Wilkings Company Baltimore, 1965, page 686) in
.under Aspergillus rugulosus Thom_':& Raper..under Aspergillus rugulosus Thom_':& Raper.
Stammen NRRL 8039 vokser på Agarnæringssubstrat mellom- 15 og 50°C, idet vekstoptimum ligger mellom 35 og 42°C. På Czapek-Dox-Agar vokser stammen NRRL 8039 heller langsomt, kulturene tilsvarer habituelt tilnærmet artsbeskrivel sen (Raper & Fennell, 1965). : 20 dager gamle kolonier er sterkt rynket og det grå, fløyel saktige luftmycel er grått, pigmentert lysebrunt til brunfiolett. Koloni-kanten blir ved lavere inkubasjonstemperaturer(£eks. 18 - 27°C) hvi t, ved ./høyere temperaturer (f.eks. 33 - 45°C) synes denne gulbrun. Baksiden av kolonien blir ved lavere inkubasjons temper aturer gulaktig, ved høyere temperaturer dannes et fiolett pigment og utskilles i agaren. Også etter 20-dagers inkubasjon på Czapek-Dox-Agar dannes ingen Cleistothecier og bare ved høyere inkubasjonstemperaturer enkle men forkrøblede konidiebærere. The strain NRRL 8039 grows on Agar nutrient substrate between 15 and 50°C, the growth optimum being between 35 and 42°C. On Czapek-Dox-Agar, the strain NRRL 8039 grows rather slowly, the cultures usually correspond approximately to the species description (Raper & Fennell, 1965). : 20-day-old colonies are strongly wrinkled and the grey, velvety aerial mycelium is grey, pigmented light brown to brownish violet. At lower incubation temperatures (e.g. 18 - 27°C), the edge of the colony becomes white, while at higher temperatures (e.g. 33 - 45°C) it appears yellowish brown. The backside of the colony becomes yellowish at lower incubation temperatures, at higher temperatures a violet pigment is formed and secreted into the agar. Also after 20-day incubation on Czapek-Dox-Agar no Cleistothecia are formed and only at higher incubation temperatures simple but stunted conidia carriers.
Betydelig hurtigere og bedre utvikles, stammen NRRL 8039 på 2% malt-dextrose-agar, hvor allerede etter 20-dagers inkubasjon ved 27°C hoved- og bifruktformene dannes i betraktelige mengder og modnes. Koloniene er høyst ganske svakt, men oftest overhode: ikke rynket.og det fLøyelsaktige luftmycel er ensartet gulaktig farget. Baksiden av kolonien er ved lavere inkubasjonstemperaturer gul og ved høyere temperaturer lysebrun. Significantly faster and better developed, the strain NRRL 8039 on 2% malt-dextrose agar, where already after 20 days of incubation at 27°C the main and secondary fruit forms are formed in considerable quantities and mature. The colonies are at most quite weak, but most often at all: not wrinkled, and the velvety aerial mycelium is uniformly yellowish in colour. The back of the colony is yellow at lower incubation temperatures and light brown at higher temperatures.
Den på 2% mali-dextrose-agar voksende stamme NRRL 8039 viser små mørkegrønne konidiehoder, som oftest opptrer i mindre grupper i og på kanten av kolonien. The strain NRRL 8039 growing on 2% mali-dextrose agar shows small dark green conidial heads, which most often appear in smaller groups in and on the edge of the colony.
Morftologien og dimensjonen av alle strukturer av bifruktformene stemmer godt overens med den nevnte artsbeskrivelse av aspergillus rugulosus. Med hensyn til hovedfruktformen består derimot noen forskjeller i forhold til artsbeskrivel sen.: således blir de kuleaktige Cleistothecier som utvikler seg i Slere lag av det fløyelsaktige luftmycel i stort antall betydelig mindre enn det er angitt for arten (50.- 80 ju i diameter i forhold til 225 - 350 /u) . Omhyllingscellene er også glassaktige, mens de.ved denne art skulle være mørkebrune. En god overensstemmelse med artsbeskrivelsen består derimot i det ar tka-rak ter is tiske trekk med de. f iolettorangerøde, linseformede 4,1 - 5,0 x 3,4 - 3,8 p store ascos<p>orer, hvis overflate-er mer eller mindre tydelig rynket og furet og hvis to parallelt anordnede ekvatorsummer er 0,4 - 0,7|U brede. ). The morphology and dimensions of all structures of the bee fruit forms agree well with the aforementioned species description of aspergillus rugulosus. With regard to the main fruit shape, however, there are some differences in relation to the species description: thus the globular Cleistothecia that develop in the Slere layer of the velvety aerial mycelium are in large numbers significantly smaller than what is indicated for the species (50.- 80 ju in diameter in compared to 225 - 350 /u) . The enveloping cells are also vitreous, whereas in this species they should be dark brown. A good agreement with the species description, on the other hand, consists in the characteristic feature of them. purple-orange-red, lenticular 4.1 - 5.0 x 3.4 - 3.8 p large ascos<p>ores, whose surface is more or less distinctly wrinkled and furrowed and whose two parallel equatorial sums are 0.4 - 0 ,7|U wide. ).
Den nye stamme NRRL 8039 lar seg dyrke på forskjellige nærings- ' substrater med de vanlige næringsstoffer, f.eks. som beskrevet i det følgende 'eksempel. The new strain NRRL 8039 can be grown on different nutrient substrates with the usual nutrients, e.g. as described in the following 'example.
De nye antibiotika SL 7810/F, SL 7810/F-II og SL 7810/F-III kan man fremstille på den måte at et flytende medium podes med en spore-eller mycel suspensjon av iammen NRRL 8039 og kulturen innkuberes i 2 - 4 dager, foretrukket 3 dager, ved 27°C. Dyrkingen skjer under aerobe betingelser, enten i overflate- eller submerskul tur. Så snart<-'en maksimal mengde av antibiotika er produsert (kan konstateres ved påvisningen av aktiviteten overfor Candida albicans) isoleres disse fra dyrkingsbuliongen etter i og for seg kjente metoder, f.eks. som beskrevet i det følgende eksempel, idet det som løsningsmiddeldestédet for etyl acetat også anvendes andre organiske løsningsmidler, som f.eks. butylacetat eller butanol. The new antibiotics SL 7810/F, SL 7810/F-II and SL 7810/F-III can be prepared in such a way that a liquid medium is inoculated with a spore or mycelial suspension of iammen NRRL 8039 and the culture is incubated for 2 - 4 days, preferably 3 days, at 27°C. Cultivation takes place under aerobic conditions, either in surface or submersible conditions. As soon as <-'a maximum quantity of antibiotics has been produced (can be ascertained by the detection of the activity against Candida albicans) these are isolated from the culture broth according to methods known per se, e.g. as described in the following example, where other organic solvents are also used as the solvent substitute for ethyl acetate, such as e.g. butyl acetate or butanol.
For separasjon fra fettaktige følgestoffer fra den rå ekstrakt kan det som første operasjon gjennomføres en filtrering over en kiselgel-kolonne, idet omtrent den 4 - 5-dobbelte vek.tmengde kiselgel, regnet på ekstraktmengden, anvendes. Elueringen skjer med kloroform-metanolblandinger (se det etterfølgende eksempel). For separation from fatty secondary substances from the raw extract, a filtration over a silica gel column can be carried out as a first operation, with approximately the 4-5 times the amount of silica gel by weight, calculated on the amount of extract, being used. The elution takes place with chloroform-methanol mixtures (see the following example).
En annen fremgangsmåte kan f .eks. gjennomføres på følgende måte: Fettaktige følgestoffer og andre biprodukter fraskilles ved passende ' fel1ingsreaksjoner. For dette utgnis etylacetatekstrakten med petroleter, idet de overfor kandidå/albikans aktive antibiotika forblir i den uløselige del. Denne rest oppslemmes imetanol og etter frafil trering av uoppløselige inaktive andeler inndampes filtratet i vakuum. Dette material løses i metanol og den klare løsning til.dryppes til den^.lO-dobbel te mengde etylacetat. Fellingen inneholder SL 7810/F, SL 7810/F-II og SL 7810/F-III ved siden av inaktive følgestoffer. Den ytterligere rensing og oppdeling av komponentene fra blandingen skjer nå ved hjelp av i;.og for seg kj~ente kromatografiske metoder, hvorved gelkromatografering på . "Séphadex LH 20" og kromatografering pa kiselgel egner seg meget godt. Disse metoder anvendes i kombinasjon og for rensing av de enkelte komponenter. Således kromatograferes f.eks. et av de ovenfor beskrevne råprodukter først på "Séphadex LH 20" med metanol, idet de tre antibiotika ved elueringen opptrer i de samme fraksjoner. Another method can e.g. carried out in the following way: Fatty by-products and other by-products are separated as appropriate ' fel1ing reactions. For this, the ethyl acetate extract is rubbed with petroleum ether, as the antibiotics active against candida/albicans remain in the insoluble part. This residue is slurried in imethanol and, after filtering off insoluble inactive parts, the filtrate is evaporated in a vacuum. This material is dissolved in methanol and the clear solution is added dropwise to the 10-fold amount of ethyl acetate. The precipitate contains SL 7810/F, SL 7810/F-II and SL 7810/F-III alongside inactive by-products. The further purification and separation of the components from the mixture now takes place using chromatographic methods known per se, whereby gel chromatography on . "Séphadex LH 20" and chromatography on silica gel are very suitable. These methods are used in combination and for cleaning the individual components. Thus, e.g. chromatographed one of the above-described raw products first on "Séphadex LH 20" with methanol, the three antibiotics appearing in the elution in the same fractions.
De forenede aktive fraksjoner kromatograferes deretter på kiselgel, kornstørrelsen 0,06 - 0,2 mm, For eluéring kan anvendes enten kloroform, som tilsettes stigende mengder metanol, men også'en kloroform-metanol-vannblanding (80 : 17,5 : 2) kan anvendes gjennomgående. Tilsvarende R^-verdiene i tynnskiktkromatogrammet erholdes The combined active fractions are then chromatographed on silica gel, grain size 0.06 - 0.2 mm. Either chloroform can be used for elution, to which increasing amounts of methanol are added, but also a chloroform-methanol-water mixture (80 : 17.5 : 2) can be used throughout. The corresponding R^ values in the thin-layer chromatogram are obtained
ved elueringen fra kiselgelkolonnene først SL 7810/F-III,in the elution from the silica gel columns first SL 7810/F-III,
deretter SL 7810/F-II og til sist SL 7810/F. For oppdeling av blandingsfraksjoner, spesielt SL 7810/F-II og SL 7810/F-III kromatograferes eventuelt lere ganger på den 250- til 400- .i dobbelte mengde kiselgel. De overfor kandida albikans aktive og i tynnskiktkromatogram rene fraksjoner tilsvarer R^-verdiene for angjeldende antibiotika. then SL 7810/F-II and finally SL 7810/F. For separation of mixture fractions, especially SL 7810/F-II and SL 7810/F-III, chromatograph if necessary several times on the 250- to 400-.in double amount of silica gel. The fractions active against Candida albicans and pure in thin-layer chromatograms correspond to the R^ values for the antibiotics in question.
Fremgangsmåten b) lar seg også gjennomføre etter i og for segProcedure b) can also be carried out in and of itself
kjente metoder, f . eks . \ved_ Katalytisk hydrogenering .known methods, e.g. e.g. \ved_ Catalytic hydrogenation .
Som løsningsmiddel kommer foretrukket lavere alifatiske alkoholer, som f.eks. metanol el ler.</>etanol, på tale. Hydrogeneringen skjer, hensiktsmessig i .nøytralt eller svakt surt område, f.eks. i nærvær av svake'organiske syrer som eddiksyre. Hydrogeneringen skjer ved ■ temperaturer mellom 10 og 40°C, foretrukket mellom 20 og 25°C. Hydrogeneringen skjer hensiktsmessig under atmosfæretrykk eller" svakt forhøyet trykk, i.; nær vær av en platinakatalysator, som f.eks. PtC^, som forhydrogeneres til Pt, eller foretrukket en paladium-katal<c>ysator, som f.eks. palladium på kull eller bariumsul f at. Hydrogeneringen skjer til fullstendig hydrogenopptagning. For dette gås det ut fra at hydrogenopptagningen tilsvarer 2 mol pr. mol antibiotikum SL 7810/F, SL 7810/F-II og SL 7810/F-III med molvekt fra ca. 1000 til 1200. Ved hydrogenerte derivater av de nevnte antibiotika^kunne det i NMR-spektrum ikke påvises noen protoner som tyder på linolsyre. Preferred solvents are lower aliphatic alcohols, such as e.g. methanol or ler.</> ethanol, in speech. The hydrogenation takes place, suitably in a neutral or weakly acidic area, e.g. in the presence of weak'organic acids such as acetic acid. The hydrogenation takes place at ■ temperatures between 10 and 40°C, preferably between 20 and 25°C. The hydrogenation conveniently takes place under atmospheric pressure or slightly elevated pressure, i.e. in the presence of a platinum catalyst, such as for example PtC^, which is prehydrogenated to Pt, or preferably a palladium catalyst, such as for example palladium on charcoal or barium sulphate. The hydrogenation takes place until complete hydrogen uptake. For this, it is assumed that the hydrogen uptake corresponds to 2 mol per mol of antibiotic SL 7810/F, SL 7810/F-II and SL 7810/F-III with a molecular weight of approx. 1000 to 1200. In the case of hydrogenated derivatives of the mentioned antibiotics, no protons indicating linoleic acid could be detected in the NMR spectrum.
De erholdte hydrogenerte derivater av antibiotikaene SL 7810/F,They obtained hydrogenated derivatives of the antibiotics SL 7810/F,
SL 7810/F-II og SL 7810/F-III renses om nødvendig på i og for seg kjent måte. Foretrukket egner seg for dette kromatogragering på "Séphadex LH 20" (eluering med metanol) og/eller kromatografering på finkornet kiselgel, idet det hensiktsmessig elueres med kloroform metanolblandinger (9:1) til (3:1) eller med kloroform-metanolblandinger under tilsetning av en liten mengde vann. SL 7810/F-II and SL 7810/F-III are cleaned if necessary in a manner known per se. Chromatography on "Séphadex LH 20" (elution with methanol) and/or chromatography on fine-grained silica gel is preferably suitable for this, as it is suitably eluted with chloroform-methanol mixtures (9:1) to (3:1) or with chloroform-methanol mixtures while adding of a small amount of water.
Den foreliggende oppfinnelse omfatter også gjæringsløsninger, som erholdes ved dyrking av en stamme av slekten aspergillus rugulosus Thorn & Raper som produserer et av antibiotikaene-; SL 7810/F, SL 7810/F II og/eller SL 7810/F-III. The present invention also includes fermentation solutions, which are obtained by cultivating a strain of the genus aspergillus rugulosus Thorn & Raper which produces one of the antibiotics-; SL 7810/F, SL 7810/F II and/or SL 7810/F-III.
De nye antibiotika og deres tetrahydroderivater'viser interessante farmakologiske egenskaper. The new antibiotics and their tetrahydroderivatives show interesting pharmacological properties.
For biologisk karakterisering av antibiotikaene > SL 7810/F, SL 7810/F-II, SL 7810/F-III og deres tetrahydroderivater tjener deres virkningsspektrum. Mens samtlige forbindelser ved de vanlige testbetingelser er uvirksomme mot grampositive og gram-negative bakterier som f.eks. staphylokokkus aureus og escherichia koli, viser de utpregende virkninger mot gjær- og sopp-arter. For biological characterization of the antibiotics > SL 7810/F, SL 7810/F-II, SL 7810/F-III and their tetrahydroderivatives serve their spectrum of action. While all compounds under the usual test conditions are inactive against gram-positive and gram-negative bacteria such as e.g. staphylococcus aureus and escherichia coli, show distinct effects against yeast and fungal species.
I den etterfølgende tabell er de minste hemmende konsentrasjonerIn the following table are the minimum inhibitory concentrations
mot forskjellige gjær- og sopp-arter oppført.. Den for dette anvendte .rekke-f ortynningsprøve ble gjennomført på kjent måte med de angitte teststammer. Inkubasjonen ved rekke-fortynningstesten skjedde i et mal tekstrakt-medium (2%) med pH-4,4 - 4,7 ved 27 til 37°C alt etter temperaturoptimum for den angjeldende teststamme. Bedømmelsen skjedde etter 72 timers inkubasjonstid. Teststammene against various yeast and fungal species listed. The serial dilution test used for this was carried out in a known manner with the indicated test strains. Incubation in the serial dilution test took place in a malt extract medium (2%) with pH 4.4 - 4.7 at 27 to 37°C depending on the temperature optimum for the test strain in question. The assessment took place after 72 hours of incubation. The test strains
er deponert i mykoteket i firma Sandoz og står der til disposisjon. is deposited in the mycotheque of the company Sandoz and is available there.
Som det fremgår av de ovenstående resultater (MIC-verdier) viser As can be seen from the above results (MIC values) show
de nevnte forbindelser en virksomhet mot bestemte sopparter,the aforementioned compounds an activity against specific fungal species,
spesielt mot candida albicans. Virkningen mot candida albicans kan også bestemmes in vivo ved følgende forsøk:. especially against candida albicans. The effect against candida albicans can also be determined in vivo by the following tests:
1. Eksperimentell tarminfeksjon med candida albikans1. Experimental intestinal infection with candida albicans
A 124 i hunrimusA 124 in female mice
.--.Ved denne metode forstyrres likevekten i den'normale tarmflora til beste for peroralt tilført candida albicans^124. Ved den bredspektrum-antibiotiske behandling opptrer undertrykkelse av den bakterielle flora og dermed til selektiv formering av candida-s-.opp. ar tene i tarmen i hunnmusene. De i tarmen dyrkede candida-organismer utskilles med avføringen (, kimtall 10 4 /g avføring). regelmessig i løpet av minst tre uker. .--.With this method, the equilibrium in the normal intestinal flora is disrupted to the advantage of orally administered candida albicans^124. With the broad-spectrum antibiotic treatment, suppression of the bacterial flora occurs and thus to the selective reproduction of candida-s-.opp. scars in the intestine of the female mice. The Candida organisms cultivated in the intestine are excreted in the faeces (, bacterial count 10 4 /g faeces). regularly during at least three weeks.
Material og metodeMaterial and method
Forsøksdyr:Laboratory animals:
Det anvendes hunn-NMRI-mus som var foret ad libitum med musepellets og som hadde en vekt på 18 - 24 g. I drikkevannet var det tilsatt Female NMRI mice are used which were fed ad libitum with mouse pellets and which had a weight of 18 - 24 g. In the drinking water it was added
1 mg/ml streptomycin.1 mg/ml streptomycin.
5l22!syl££i!22_22_in^'e'<:s jon • 5l22!syl££i!22_22_in^'e'<:s ion •
Tarminfeksjonen av hunnmusene med candida albicans ble gjennomførtThe intestinal infection of the female mice with candida albicans was carried out
på følgende.måte:in the following manner:
Fra en i flytende nitrogen oppbevart og opptint ampulle ble det fremstilt en fortynning som inneholdt 5 x 10 7 kimer i 0,25 ml. From a vial stored in liquid nitrogen and thawed, a dilution containing 5 x 10 7 germs in 0.25 ml was prepared.
Det ble så ° i det. enkelte tilfelle tilført .5 x 10 7 kim pr. mus peroralt Etter infeksjonen ble dyrene fordelt enkeltvis i bur (6 eller 10 It became so ° in it. in some cases added .5 x 10 7 germs per mice orally After the infection, the animals were distributed individually in cages (6 or 10
dyr som holdes enkeltvis utgjør et kollektiv). animals kept individually constitute a collective).
!5i!I!;!:^Iikestemmelse_i avfØ ri n9 (KZ/g avføring) :!5i!I!;!:^Iikestemmelmes_in excrement n9 (KZ/g excrement) :
Forsøksdyrene ble den 6. dag - henhv. på det tidspunkt hvor kimtallet i avføringen skal bestemmes,vsatt i sterile makrolonbur og avføring i 4 timer ble samlet og.veid. Deretter ble det i en NaCl-løsning fremstilt en suspensjon på 10 — 1 og fortynnet til 10 —7. Deretter ble kimene telt i platemetoden med saboraud-agar, idet det pr. ml SAB-agar ble tilsatt 50 yug streptomycin. Ved streptomycin-tilsetningen i agaren ble veksten av de øvrige tarmkimer utskjaltet og det ble på agarplaten erholdt en renkultur av sopparten candida albicans. The experimental animals were on the 6th day - respectively at the time when the germ count in the faeces is to be determined, placed in sterile macrolon cages and faeces for 4 hours were collected and weighed. A suspension of 10 - 1 was then prepared in a NaCl solution and diluted to 10 -7. The germs were then counted in the plate method with Saboraud agar, as per ml of SAB agar was added with 50 µg of streptomycin. When streptomycin was added to the agar, the growth of the other intestinal germs was suppressed and a pure culture of the fungus Candida albicans was obtained on the agar plate.
Dosering_og behandlingsmåte:Dosage_and method of treatment:
Den høyeste tilførte dose av enttestsubstans retter seg generelt etter dosen tolerata maksima. Den utgjør høyst 300 mg/kg kroppsvekt for mus. En ytterligere dosering utgjør 100 mg/kg kroppsvekt. The highest administered dose of the test substance generally follows the dose tolerata maxima. It amounts to a maximum of 300 mg/kg body weight for mice. A further dosage amounts to 100 mg/kg body weight.
Disse daglige tilførselsdoser, tilføres, begynnende med dagen for infeksjonen, peroralt i 5 dager. Dagsdosene fordeles på hver tre enkeltdoser og tilføres peroralt i et volum på 0,25 ml (ved vann-uoppløselige substanser tjener 0,2% KMC + 0,2% "Tween 80" som suspensjonsformidlere) til 20 g mus. These daily supply doses are administered orally for 5 days, beginning on the day of the infection. The daily doses are divided into three single doses and administered orally in a volume of 0.25 ml (for water-insoluble substances, 0.2% KMC + 0.2% "Tween 80" serve as suspension agents) to 20 g mice.
Hvert infeksjonsforsøk ble komplettert ved medføring av 10 kontrolldyr, som erholdt en standardsubstans. Som standardsubstans ble i dette infeksjonsforsøk tilført "Nystatin" i dagsdoser på 300 og 100 mg/kg kroppsvekt peroralt. Tilførselen av disse doser skjedde som ved testsubstansen. Ved en p.o. tilført dose på 300 mg/kg kroppsvekt "Nystatin" er det en dag etter avsluttet behandling ikke mulig å påvise noen kimer av Candida albicans i avføringen, mens 100 mg/kg kroppsvekt "Nystatin" bevirker en kimreduksjon på å minst to tierpotenser, i noen tilfeller også allerede en fullstendig eliminering av disse kim. Each infection trial was completed by including 10 control animals, which received a standard substance. As a standard substance, "Nystatin" was added in daily doses of 300 and 100 mg/kg body weight orally in this infection experiment. The supply of these doses took place as with the test substance. At a p.o. given a dose of 300 mg/kg body weight "Nystatin", one day after the end of treatment it is not possible to detect any germs of Candida albicans in the faeces, while 100 mg/kg body weight "Nystatin" causes a germ reduction of at least two tier powers, in some cases also already a complete elimination of these germs.
For hver dosering foreligger en gruppe på 6 dyr hver, som holdes enkeltvis i bur. 6 ytterligere dyr erholder som alle andre også streptomycin i drikkevannet og løsningsmidlet uten substans som kontroll. Dyrene holdes likeledes enkeltvis. For each dosage there is a group of 6 animals each, which are kept individually in cages. 6 additional animals, like all others, also receive streptomycin in the drinking water and the solvent without substance as a control. The animals are also kept individually.
Bestemmelse og bedømmelse:Determination and assessment:
Ved bestemmelsen av.et slikt forsøk ble kimtallreduksjoner henhv. kim-elimineringer bestemt og sammenliknet med de ubehandlede kontrolldyr henhv. sammenliknet' med standardsubstansenes kontrolldyr When determining such an experiment, reductions in germ counts were respectively germ eliminations determined and compared with the untreated control animals or compared' with the standard substances' control animals
og beregnet i prosent.and calculated as a percentage.
2. Vaginal Candida albicans- infeksjon i rotter2. Vaginal Candida albicans infection in rats
(stamme Al24)(stem Al24)
Den vaginale mykose i hunnrotter utgjør en slimhud-infeksjon. Ved ovariektomi og etterfølgende hormon til setning indusert' varig brunst i den kjønnsmodne rotte kan det frembringes en eksperimentell vaginal mykose. Denne lokale infeksjon omfatter omtrent bare de ytterste overf1ateskikt av vaginal slimhuden. Véd avstrykninger kan dens forløp kontrolleres nøye. Den gode reproduserbarhet og det ensartede forløp av infeksjonene oppfyller alle de krav som er forutsetningen for letingen etter et nytt virkestoff. Vaginal mycosis in female rats is a mucosal infection. An experimental vaginal mycosis can be produced by ovariectomy and subsequent hormone administration to induce permanent estrus in the sexually mature rat. This local infection covers approximately only the outermost surface layers of the vaginal mucosa. With smears, its progress can be carefully controlled. The good reproducibility and the uniform course of the infections meet all the requirements that are the prerequisite for the search for a new active ingredient.
Forsøksdyr^Laboratory animals^
Det ble anvendt wistar-ftunnrotter med en vekt på ca. 200 g. Dyrene fikk mat og vann ad libitum. For hver forsø.ksgruppe ble Wistar rats with a weight of approx. 200 g. The animals received food and water ad libitum. For each experimental group,
det anvendt 10 dyr.10 animals were used.
Forberedelse og gjennomføring av infeksjonen:Preparation and implementation of the infection:
Under "N.embutal-"-narkose ble dyrene ovari ek tome. I'; En dag etter ovariektomien ble 100^ug østradiol tilført i /en_ mengde på 0,5 ml sesamolje subkutantpr. rotte. En uke etter hormontilsetningen befannt dyrene seg i østrustil stand idet dette lett lot seg påvise ved hjelp av avstrykninger og metylenblåttfarging i mikroskop.. Under "N.embutal" anesthesia, the animals were ovariectomized. IN'; One day after the ovariectomy, 100 µg estradiol was added subcutaneously in a quantity of 0.5 ml sesame oil. rat. One week after the hormone addition, the animals were in an estrus state, as this could easily be demonstrated using smears and methylene blue staining under a microscope.
I denne tilstand av varigc:bruns t kan det i vaginal innholdet knapt finnes leu.coeyter, idet dette er en fordel for manifestas jon' av en infeksjon, da f renimedkimer som trenger inn i vagina ikke -med en gapg fagocyteres av leucocytene. 7 dager etter ovariektomien skjer infeksjonen. Med en tuberkulin-sprøyte og slundsonde tilføres candida albicanskimer i en mengde på 0,05 ml eller 10^ kimer pr. rotte intravaginal. In this state of varigc:browns t there can hardly be leucocytes in the vaginal contents, as this is an advantage for the manifestation of an infection, as germs that penetrate the vagina are not phagocytosed by the leucocytes. 7 days after the ovariectomy, the infection occurs. With a tuberculin syringe and throat probe, Candida albicans germs are added in a quantity of 0.05 ml or 10^ germs per rat intravaginally.
Fremstillingen av inokulum skjer etter vanlige metoder og oppbevaresThe production of inoculum takes place according to usual methods and is stored
1 flytende nitrogen. Kimene anvendes direkte fra etter hurtig opptining for infeksjonen. Forløpet av den lokale vaginalmykose kontrolleres 3., 7., 10., 15. og 20. dag etter infeksjonen. Vaginal innhold strykes ut med en platinaring på str eptomycinholdig '" sabouraud-agar plate (20 ^ug/ml) . Parallelt fremstilles med vaginal-innhold avstrykninger som farges for grampåvisning og undersøkes under mikroskopet for tilstedeværelse av seudomycel. Etter 2 dagers dyrking av SAB-platene (rør) ved 30°C bedømmes disse semi-kvantitativt. Alt etter veksten av candida albicanskolonier. kan man tale om en høygradig (hurtigvekst), måtelig (tellbare kolonier) og lavgradig (enkelt-vekst av kolonier av candida albicans) infeksjon. 1 liquid nitrogen. The germs are used directly from after rapid thawing for the infection. The course of the local vaginal mycosis is checked on the 3rd, 7th, 10th, 15th and 20th days after the infection. Vaginal contents are smeared with a platinum ring on str eptomycin-containing Sabouraud-agar plate (20 µg/ml). In parallel, smears are prepared with vaginal contents which are stained for Gram detection and examined under the microscope for the presence of pseudomycelium. After 2 days of cultivation of SAB plates (tubes) at 30°C, these are assessed semi-quantitatively. Depending on the growth of candida albicans colonies, one can speak of a high-grade (rapid growth), moderate (countable colonies) and low-grade (single-growth of colonies of candida albicans) infection .
Beihandl ing_og_doseri ng_:Handling_and_dosing_:
Den lokale behandling av den eksperimentelle vagina-kadidasis begynner 24 timer etter infeksjonen. Det gis intravaginal-terapi 2 ganger daglig (formiddag og ettermiddag) 5 etterfølgende dager. The local treatment of the experimental vaginal caddisiasis begins 24 hours after the infection. Intravaginal therapy is given twice a day (morning and afternoon) for 5 consecutive days.
Som enkeltdose av en testsubstans tilføres i det enkelte tilfelleAs a single dose of a test substance is administered in the individual case
0,5 ml av en 1,5% og en 5% salve pr. rotte.0.5 ml of a 1.5% and a 5% ointment per rat.
Substansene forarbeides i en blanding av 2 deler•polyetylenglykolThe substances are processed in a mixture of 2 parts • polyethylene glycol
300 og 1 del polyetylenglykol 1500 "salvebasis" og tilføres intravaginalt. 300 and 1 part polyethylene glycol 1500 "ointment base" and administered intravaginally.
Hvert infeksjonsforsøk ble komplettert ved medføring av en gruppeEach infection trial was supplemented by the inclusion of a group
på 10 dyr som kontrolldyr, som fikk en kjent standardsubstans. Som standardsubstans ble anvendt"Nystatin" som'1,5% salve henhv. alternativt "Clotrimazol" som 1,5% salve. 10 ytterligere dyr fikk salvebasis uten substans og en gruppe på 10 dyr tjener som infeksjons-kontrol1. on 10 animals as control animals, which received a known standard substance. As a standard substance, "Nystatin" was used as a 1.5% ointment or alternatively "Clotrimazole" as 1.5% ointment. 10 additional animals received ointment base without substance and a group of 10 animals serves as infection control1.
De nye antibiotika og deres tetrahydroderivater egner seg derfor for behandling av de ved Candida albicans fremkalte sykdommer. The new antibiotics and their tetrahydroderivatives are therefore suitable for the treatment of diseases caused by Candida albicans.
Vanlig oppnås tilfredsstillende resultater,.med en daglig dose på omtrent 100 til 1500 mg. Denne dose kan om nødvendig tilføres i Satisfactory results are usually obtained with a daily dose of approximately 100 to 1500 mg. If necessary, this dose can be administered i
2 til 3 porsjoner på 30 til 750 mg.2 to 3 servings of 30 to 750 mg.
Som legemiddel kan de nye antibiotika og deres tetrahydroderivater tilføres alene eller i passende preparatform sammen med uorganiske eller organiske, farmakologisk indifferente hjelpestoffer. F.eks. anvendes de som b'estanddel av en salve, idet konsentrasjonen i salven kan utgjøre ca. 5 til 130 mg virkestoff pr. gram salve. Andre preparatformer er tablettei; kapsler og løsninger. As medicine, the new antibiotics and their tetrahydroderivatives can be administered alone or in suitable preparation form together with inorganic or organic, pharmacologically indifferent excipients. E.g. they are used as a component of an ointment, as the concentration in the ointment can amount to approx. 5 to 130 mg of active ingredient per gram of ointment. Other forms of preparation are tablets; capsules and solutions.
I de etterfølgende eksempeler som skal illustrere oppfinnelsen,In the following examples to illustrate the invention,
er alle temperaturangivelser i grader celcius. Alle angitte forhold er på volumbasis. All temperature indications are in degrees Celsius. All stated ratios are on a volume basis.
Eksempel 1:Example 1:
En sporesuspensjon som tjener for poding av næringsløsningen, fremstilles av en kultur av stammen NRRL 8039. For dette podes 200 ml næringsagar med følgende sammensetning: A spore suspension that serves for inoculation of the nutrient solution is produced from a culture of strain NRRL 8039. For this, 200 ml of nutrient agar with the following composition is inoculated:
Etter 10 'dagers inkubasjon ved- 2.7°C suspenderes de dannede sporer After 10 days of incubation at 2.7°C, the formed spores are suspended
i 200 ml sterilt vann og et ståi-gjæringskar som inneholder 50'liter av følgende næringsløsning, podes dermed: in 200 ml of sterile water and a standing fermentation vessel containing 50 liters of the following nutrient solution, inoculate the following:
B så vel som 10% polypropylenglykol 2020 (Chem. Werke Huls, Mari B as well as 10% polypropylene glycol 2020 (Chem. Werke Huls, Mari
BRD) BRD)
1 liter av-ionisert vann.1 liter of de-ionized water.
Denne gjæringsbeholder ble inkubert under omrøring i 42 timer ved 27°C, idet luftingen utgjorde 1 liter luft/min/liter medium, ved en omrøringsfasthet på 250 omdr./min.. This fermentation container was incubated with stirring for 42 hours at 27°C, with the aeration amounting to 1 liter of air/min/liter of medium, at a stirring speed of 250 rpm.
Denne kultur tjener som forkultur for 500 liter.^gjæringskaret -medThis culture serves as a pre-culture for 500 litres.^the fermentation vessel -with
den samme næringsløsning som ble inkubert dermed i 66 timer ved 27°C. Beluftningen utgjorde på nytt 1 liter luft/min./liter medium, men omrøringshastigheten utgjorde bare 150 omdr./min. Etter 66 timers inkubasjonstid ble dyrkingsbuliongen høstet og opparbeidet på 'følgende måte: 450 liter dyrkingsbuliong ble innstilt til pH 7,0 med.2 N saltsyre, tilsatt 500 liter etylacetat og homogenisert med en "Dispax"-reaktor. Deretter ble den organiske fase skilt fra buliongen.med"separator og vasket med 50 liter vann. Dette ekstråksjonsskritt the same nutrient solution which was thus incubated for 66 hours at 27°C. The aeration was again 1 liter of air/min./liter of medium, but the stirring speed was only 150 rpm. After 66 hours of incubation, the culture broth was harvested and worked up as follows: 450 liters of culture broth were adjusted to pH 7.0 with 2 N hydrochloric acid, 500 liters of ethyl acetate were added and homogenized with a "Dispax" reactor. The organic phase was then separated from the broth using a separator and washed with 50 liters of water. This extraction step
ble gjentatt to ganger. De erholdte 3 ekstrakter ble forenet og inndampet til tørrhet under vakuum (vannringpumpe)' ved 20 til 4\0°C. was repeated twice. The 3 extracts obtained were combined and evaporated to dryness under vacuum (water ring pump) at 20 to 40°C.
Dette material fraksjoneres på den 4-dobbelte mengde kiselgel'"Merk 60" (kornstørrelse 0,063 - 0,20 mm)..Eluatene.med kloroform + 10% metanol .og kloroform + 20% metanol inneholdt inaktive følge-substanser. Kromatografikolonnen ble deretter eluert med kloroform-metanol (1 : 1) og fraksjonene med aktivitet overfor Candida albicans''ble forenet og inndampet til .tørrhet. Ved kromatograf er ing av 16,4 g aktivt material på;1 kg kiselgel "Merk 60" (kornstørrel se 0,063 - 0,20 mm) og eluering med kloroform + 20% metanol ble det først ebholdt blandingsfraksjoner av SL 7810/F-II og SL 7810/F-III. Den^^etterfølgende eluering med klorofor m + 30% metanol gav fraksjoner This material is fractionated on the 4-fold amount of silica gel "Mark 60" (grain size 0.063 - 0.20 mm). The eluates with chloroform + 10% methanol and chloroform + 20% methanol contained inactive secondary substances. The chromatography column was then eluted with chloroform-methanol (1:1) and the fractions with activity against Candida albicans were combined and evaporated to dryness. By chromatographing 16.4 g of active material on 1 kg of silica gel "Mark 60" (grain size see 0.063 - 0.20 mm) and elution with chloroform + 20% methanol, mixed fractions of SL 7810/F-II were first obtained and SL 7810/F-III. The subsequent elution with chloroform + 30% methanol gave fractions
-med SL 7810/F som hovedkomponent. Etter gel filtrering av SL 7810/F fraksjoner (8,1 g) på 1,2 kg "Séphadex LH 20" med metanol ble SL 7810/F utvunnet i tilnærmet ren form. Dette preparat ble åsøst i aceton under oppvarming og den klare løsning inndampes til é/4 av volumet. Etter 3 timers henstand ved -15°G frafiltreres det dannede -with SL 7810/F as main component. After gel filtration of SL 7810/F fractions (8.1 g) on 1.2 kg "Séphadex LH 20" with methanol, SL 7810/F was recovered in almost pure form. This preparation was dissolved in acetone while heating and the clear solution was evaporated to 1/4 of the volume. After standing for 3 hours at -15°G, the formed is filtered off
bunnfall og tørres etter vasking med aceton og eter i 24 tåmer ved romtemperatur over & 2®5 ^ høyvakuum. SL 7810/F erholdes som fargeløst pulver med smeltepunkt 160 - 163°C (spalting). precipitate and dried after washing with acetone and ether for 24 hours at room temperature above & 2®5 ^ high vacuum. SL 7810/F is obtained as a colorless powder with a melting point of 160 - 163°C (decomposition).
Ved fortsatt kromatografering av de aktive blandingsfraksjoner av SL 7810/F-II og SL 7810/F-III .fra den ovennevnte kiselgelkromato-grafering kunne de to atatibiotika isoleres. For dette løses materialet (2,5 g) i kloroform + 20% metanol og løsningen ble absorbert på en kolonne med 1 kg kiselgel, som var behandlet med den samme løsningsmiddelblanding. Elueringen skjedde gjennomgående med kloroform + 20% metanol, idet først overveiende SL 7810/F-III elueres og deretter overveiende;SL 7810/F-II. For utvinning av rent SL 7810/F-III løses de inndampede blandingsfKaksjoner (0,75 g) som inneholder dette antibiotikum, i kloroform + 10% metanol og løsningen, adsorberes analogt med det som tidligere er beskrevet på 1 kg kiselgel "Merk". Elueringen ved kloroform + 10% metanol til 15% metanol gav det sterkt anrikede material av SL 7810/F-III. Etter Éjelf il trering (0,55 g) på 500 g "Séphadex LH 20" med metanol erholdes rent SL 7810/F-III: Fargeløst amorft pulver med smeltepunkt 164 - 168°C (spalting). By continued chromatography of the active mixture fractions of SL 7810/F-II and SL 7810/F-III from the above-mentioned silica gel chromatography, the two atatibiotics could be isolated. For this, the material (2.5 g) is dissolved in chloroform + 20% methanol and the solution was absorbed on a column with 1 kg of silica gel, which was treated with the same solvent mixture. The elution took place throughout with chloroform + 20% methanol, with first predominantly SL 7810/F-III being eluted and then predominantly SL 7810/F-II. For the extraction of pure SL 7810/F-III, the evaporated mixed fractions (0.75 g) containing this antibiotic are dissolved in chloroform + 10% methanol and the solution is adsorbed analogously to what was previously described on 1 kg of silica gel "Mark". The elution with chloroform + 10% methanol to 15% methanol gave the highly enriched material of SL 7810/F-III. After Éjelf il trituration (0.55 g) of 500 g "Séphadex LH 20" with methanol, pure SL 7810/F-III is obtained: Colorless amorphous powder with melting point 164 - 168°C (decomposition).
Fjor isolering av SL 7810/F-II løses blandingsf raks j onene (l,7.£.§) som inneholder SL 7810/F-II i kloroform + 20% metanol og denne løsning adsorberes på en medden samme løsnåmgsmiddelblancling behandlede kolonne med 1 kg kiselgel. Elueringen med kloroform + 20% metanol gir steEkt anrikede fraksjoner av SL 7810/F-II. Den etterfølgende gelfiltreringe(1,2 g) på 1,2 kg ^Séphadex LH 20" med metanol gir rent SL 7810/F-II, et amorft pulver med smeltepunkt '158 / 160°C (spalting). Before isolating SL 7810/F-II, the mixture fractions (1,7.£.§) containing SL 7810/F-II are dissolved in chloroform + 20% methanol and this solution is adsorbed on a column treated with the same solvent blanking with 1 kg silica gel. The elution with chloroform + 20% methanol gives strongly enriched fractions of SL 7810/F-II. The subsequent gel filtration (1.2 g) of 1.2 kg ^Séphadex LH 20" with methanol gives pure SL 7810/F-II, an amorphous powder with melting point '158 / 160°C (decomposition).
Eksempel 2: Tetrahydro- SL 7810/ FExample 2: Tetrahydro-SL 7810/ F
En suspensjon av 1 g palladiumkull (10% Pd) i 250 ml alkohol eller 250 ml alkohol-iseddik (l : 1) forhydrogeneres i 30 min. Deretter tilsettes en løsning av 5 g SL 7810/F i 50 ml alkohol-iseddik A suspension of 1 g of palladium charcoal (10% Pd) in 250 ml of alcohol or 250 ml of alcohol-glacial vinegar (1:1) is prehydrogenated for 30 min. A solution of 5 g of SL 7810/F in 50 ml of alcohol-glacial vinegar is then added
(1 : 1). Etter 3 timers hydrogenering ved 20°C under atmosfæretrykk fraskilles katalysatoren ved filtrering og det klare filtrat inndampes i vakuum. Inndampningsresten løses i kloroform-metanol (1 : 1). After 3 hours of hydrogenation at 20°C under atmospheric pressure, the catalyst is separated by filtration and the clear filtrate is evaporated in a vacuum. The evaporation residue is dissolved in chloroform-methanol
(l : 1), med denne løsning impregneres 10 g kiselgel 60 "Merk" (l : 1), with this solution impregnate 10 g of silica gel 60 "Mark"
(kornstørrelse 0,063 - 0,20 mm) og dette innføres på en kolonne medj500 g kiselgel. Den etterfølgende eluering med kloroform-metanol (4 : l) gir det amorfte hydrogeneringsprodukt som fargeløst pulver med smeltepunkt 173 - 178°C (spalting). Preparatet krystalliseres fra etanol-vann (95 : 5). Smeltepunkt etter tørring i 3 timer i høyvakuum med 50°C: 190 - 205°C / 210 - 212°C (under spalting). (grain size 0.063 - 0.20 mm) and this is introduced onto a column with 500 g of silica gel. The subsequent elution with chloroform-methanol (4:1) gives the amorphous hydrogenation product as a colorless powder with a melting point of 173 - 178°C (decomposition). The preparation is crystallized from ethanol-water (95:5). Melting point after drying for 3 hours in high vacuum at 50°C: 190 - 205°C / 210 - 212°C (under decomposition).
Eksempel 3: Tetrahydro- SL 7810/ F- IIExample 3: Tetrahydro-SL 7810/F-II
, Fremstillingen skjer analogt med eksempel 2, idet det som utgangs-material anvendes antibiotikum SL 7810/F-II. , The preparation takes place analogously to example 2, in that antibiotic SL 7810/F-II is used as starting material.
Eksempel 4: Tetrahydro- SL 7810/ F- IIIExample 4: Tetrahydro-SL 7810/F-III
Fremstillingen skjer analogt med eksempel1 2, idet det som utgangs-material anvendes antibiotikum SL 7810/F-III, hydrogeneringsvarig-heten var 4 timer og som løsimingsmiddelblanding for kromatograferingen på kiselgel ble det anvendt kloroform-metanol-vann (80 : 17,5 : 2). The preparation takes place analogously to example 1 2, in that the antibiotic SL 7810/F-III is used as the starting material, the hydrogenation duration was 4 hours and as the solvent mixture for the chromatography on silica gel chloroform-methanol-water (80 : 17.5 : 2).
Claims (4)
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CH1342574 | 1974-10-07 | ||
CH1655674A CH606069A5 (en) | 1974-12-12 | 1974-12-12 | Antibiotic SL 7810 F and their tetrahydro derivs |
CH171375A CH616705A5 (en) | 1975-02-12 | 1975-02-12 | Process for the preparation of the metabolites SL 7810/F-II and SL 7810/F-III. |
CH436475 | 1975-04-07 | ||
CH967975 | 1975-07-24 |
Publications (1)
Publication Number | Publication Date |
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NO753291L true NO753291L (en) | 1976-04-08 |
Family
ID=27508997
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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NO753291A NO753291L (en) | 1974-10-07 | 1975-09-29 |
Country Status (12)
Country | Link |
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JP (1) | JPS5195189A (en) |
AU (1) | AU508075B2 (en) |
BE (1) | BE834289A (en) |
DK (1) | DK438475A (en) |
FI (1) | FI752696A (en) |
FR (1) | FR2287235A1 (en) |
GB (1) | GB1527494A (en) |
IE (1) | IE43592B1 (en) |
IL (1) | IL48248A0 (en) |
NL (1) | NL7511649A (en) |
NO (1) | NO753291L (en) |
SE (1) | SE7510899L (en) |
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US4293491A (en) * | 1979-12-13 | 1981-10-06 | Eli Lilly And Company | Derivatives of A-30912H nucleus |
US4304716A (en) * | 1979-12-13 | 1981-12-08 | Eli Lilly And Company | S 31794/F-1 Nucleus |
US4293485A (en) * | 1979-12-13 | 1981-10-06 | Eli Lilly And Company | Derivatives of S31794/F-1 nucleus |
US4293487A (en) * | 1979-12-13 | 1981-10-06 | Eli Lilly And Company | Derivatives of A-30912D nucleus |
US4322338A (en) * | 1979-12-13 | 1982-03-30 | Eli Lilly And Company | Derivatives of A-30912B nucleus |
US4293484A (en) * | 1979-12-13 | 1981-10-06 | Eli Lilly And Company | Derivatives of A-30912H nucleus |
US4293483A (en) * | 1979-12-13 | 1981-10-06 | Eli Lilly And Company | Derivatives of A-30912A nucleus |
US4299763A (en) * | 1979-12-13 | 1981-11-10 | Eli Lilly And Company | A-30912B Nucleus |
US4293490A (en) * | 1979-12-13 | 1981-10-06 | Eli Lilly And Company | A-30912H Nuclei |
US4297277A (en) * | 1979-12-13 | 1981-10-27 | Eli Lilly And Company | Derivatives of A-30912B nucleus |
US4293482A (en) * | 1979-12-13 | 1981-10-06 | Eli Lilly And Company | A-30912A Nucleus |
US4293488A (en) * | 1979-12-13 | 1981-10-06 | Eli Lilly And Company | Derivatives of A-30912B nucleus |
US4320052A (en) * | 1979-12-13 | 1982-03-16 | Eli Lilly And Company | Derivatives of A-30912A nucleus |
US4293489A (en) * | 1979-12-13 | 1981-10-06 | Eli Lilly And Company | Derivatives of A-30912A nucleus |
US4287120A (en) * | 1979-12-13 | 1981-09-01 | Eli Lilly And Company | Derivatives of S31794/F-1 nucleus |
US4320053A (en) * | 1979-12-13 | 1982-03-16 | Eli Lilly And Company | Derivatives of A-30912D nucleus |
US4320054A (en) * | 1979-12-13 | 1982-03-16 | Eli Lilly And Company | Derivatives of A-30912H nucleus |
US4299762A (en) * | 1979-12-13 | 1981-11-10 | Eli Lilly And Company | A-30912D Nucleus |
US4293486A (en) * | 1979-12-13 | 1981-10-06 | Eli Lilly And Company | Derivatives of S31794/F-1 nucleus |
US4289692A (en) * | 1979-12-13 | 1981-09-15 | Eli Lilly And Company | Derivatives of A-30912D nucleus |
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CH79A (en) * | 1889-01-09 | Adam Sautter Johann | Movement for pocket watches with an open barrel, new gear pusher system and simplified movement fastening | |
JPS5134916B2 (en) * | 1974-03-06 | 1976-09-29 |
-
1975
- 1975-09-26 FI FI752696A patent/FI752696A/fi not_active Application Discontinuation
- 1975-09-29 NO NO753291A patent/NO753291L/no unknown
- 1975-09-29 SE SE7510899A patent/SE7510899L/en unknown
- 1975-09-29 DK DK438475A patent/DK438475A/en unknown
- 1975-10-02 GB GB40301/75A patent/GB1527494A/en not_active Expired
- 1975-10-03 NL NL7511649A patent/NL7511649A/en unknown
- 1975-10-06 JP JP50119866A patent/JPS5195189A/ja active Pending
- 1975-10-06 IE IE2187/75A patent/IE43592B1/en unknown
- 1975-10-06 IL IL48248A patent/IL48248A0/en unknown
- 1975-10-07 BE BE160769A patent/BE834289A/en unknown
- 1975-10-07 FR FR7530625A patent/FR2287235A1/en active Granted
- 1975-10-07 AU AU85523/75A patent/AU508075B2/en not_active Expired
Also Published As
Publication number | Publication date |
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DK438475A (en) | 1976-04-08 |
IL48248A0 (en) | 1975-12-31 |
FI752696A (en) | 1976-04-08 |
GB1527494A (en) | 1978-10-04 |
AU8552375A (en) | 1977-04-21 |
SE7510899L (en) | 1976-04-08 |
IE43592B1 (en) | 1981-04-08 |
FR2287235A1 (en) | 1976-05-07 |
IE43592L (en) | 1976-04-07 |
FR2287235B1 (en) | 1979-03-16 |
AU508075B2 (en) | 1980-03-06 |
NL7511649A (en) | 1976-04-09 |
BE834289A (en) | 1976-04-07 |
JPS5195189A (en) | 1976-08-20 |
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